We assessed the effects of conventional and ultra-high dose rate (UHDR) electron irradiation on behavioral and cognitive performance one month following exposure and assessed whether these effects were associated with alterations in the number of immune cells in the hippocampus using flow cytometry. Two-month-old female and male C57BL/6J mice received whole-brain conventional or UHDR irradiation. UHDR mice were irradiated with 9 MeV electrons, delivered by the Linac-based/modified beam control. The mice were irradiated or sham-irradiated at Dartmouth, the following week shipped to OHSU, and behaviorally and cognitively tested between 27 and 41 days after exposure. Conventional- and UHDR-irradiated mice showed impaired novel object recognition. During fear learning, conventional- and UHDR-irradiated mice moved less during the inter-stimulus interval (ISI) and UHDR-irradiated mice also moved less during the baseline period (prior to the first tone). In irradiated mice, reduced activity levels were also seen in the home cage: conventional- and UHDR-irradiated mice moved less during the light period and UHDR-irradiated mice moved less during the dark period. Following behavioral and cognitive testing, infiltrating immune cells in the hippocampus were analyzed by flow cytometry. The percentage of Ly6G+ CD45+ cells in the hippocampus was lower in conventional- and UHDR-irradiated than sham-irradiated mice, suggesting that neutrophils might be particularly sensitive to radiation. The percentage of Ly6G+ CD45+ cells in the hippocampus was positively correlated with the time spent exploring the novel object in the object recognition test. Under the experimental conditions used, cognitive injury was comparable in conventional and UHDR mice. However, the percentage of CD45+ CD11b+ Ly6+ and CD45+ CD11b+ Ly6G- cells in the hippocampus cells in the hippocampus was altered in conventional- but not UHDR-irradiated mice and the reduced percentage of Ly6G+ CD45+ cells in the hippocampus might mediate some of the detrimental radiation-induced cognitive effects. After the cessation of whole-brain radiotherapy with X-rays or γ-rays for brain tumors, over 50% of cancer survivors report impaired cognition . Many st6 Gy/s) or conventional IR. Simmons et al. [Following promising results in a mouse model of gastrointestinal (GI) injury using high dose rates in the 1960s and 1970s ,11,12,13s et al.  used 3-ms et al. . TherefoIrradiation and timeline of behavioral testing. Mice received whole brain conventional or UHDR irradiation as indicated in Y maze. Irradiation did not affect spontaneous alternation or entries, an activity measure, in the Y maze  = 37.85, p < 0.001) but there was no effect of treatment on activity levels over the two days of testing . However, conventional-  and UHDR-irradiated  mice did not (p = 0.0097), while conventional-  and UDHR-irradiated mice did not   C. HoweveElevated zero maze. Next, measures of anxiety were assessed in the elevated zero maze. There was a trend toward an effect of irradiation on measures of anxiety in the elevated zero maze  = 1.255, p = 0.0820) and a trend toward higher measures of anxiety in UHDR- than sham-irradiated mice  (nnett’s) D.Body weights. There was no effect of irradiation on the percentage of body weight change  in the mice  = 65.02, p < 0.0001) but there was no effect of radiation on the percentage of time spent in the light or dark compartment  = 2.947, p = 0.0885), with a trend toward a higher percentage of entries in UHDR- than sham-irradiated mice (p = 0.0830) (p = 0.0443) .  0.0830) . There w 0.0830) . When th 0.0443) , with a Fear learning and cued fear memory. During the baseline period of fear learning (prior to the first tone), there was an effect of radiation on activity levels  = 4.200, p = 0.0372)  and there was a trend toward conventional-irradiated mice moving less than sham-irradiated mice . There was no effect of radiation on the percentage of freezing during the tones (p = 0.0117) and a trend toward lower activity levels in conventional-irradiated than sham-irradiated mice (p = 0.09) (p = 0.0843) and a trend toward higher freezing levels in conventional- than sham-irradiated mice  (p = 0.0142) with lower activity levels in conventional-irradiated  and UHDR-irradiated (p = 0.0307) than sham-irradiated mice (p < 0.0001), with higher freezing levels during the tone than during the pre-tone period (p = 0.0006), with lower activity levels during the tone than pre-tone but there was no effect of radiation on cued fear memory. 0.0372) A. UHDR-ihe tones B. When a = 0.09) C. Perfornnett’s) D. There ted mice E. On daye period F. There Circadian home cage activity. Circadian activity levels were assessed over two weeks using infrared sensors in individually housed mice. During the light period, there was an effect of radiation  = 4.804, p = 0.0258) with lower activity levels in conventional-  and UHDR-irradiated  mice than sham-irradiated mice (p = 0.0258), with lower activity levels in UHDR- than sham-irradiated mice  and a trend toward lower activity levels in conventional- than UHDR-irradiated mice  (ted mice A. There nnett’s) A. Consisnnett’s) B.Flow cytometry. There was an effect of radiation on the percentage of CD45+ Ly6G+ cells in the hippocampus  = 10.82, p = 0.0005, Kruskall–Wallis) with a lower percentage in conventional-  and UHDR-irradiated  than sham-irradiated . There was no effect of radiation on the percentage of CD11b+ CD45+ cells, CD45+ cells expressing high levels of CD45, CD45+ cells expressing low levels of CD45, or 206+ CD45+ cells.radiated A mice. Ap = 0.02, two-tailed Spearman, p = 0.049, two-tailed Spearman, The percentage of CD45+ Ly6G+ cells in the hippocampus correlated positively with performance in the second trial of the grip strength test , with a lower percentage of CD45+ CD11b+ Ly6G+ cells in conventional than sham-irradiated mice  = 5.348, (Ico)-Foxn1nu) mice at 14 Gy, a dose similar to the 16 Gy in our study. Preservation of NOR in UHDR- but not conventional-irradiated mice was seen at four months, but not two months, following exposure to 10 Gy [In the current study, conventional- and UHDR-irradiated mice showed impaired novel object recognition. Consistent with these findings, in the only study that involved NOR testing at one-month post-exposure, comparable to the timeline of our study, NOR was impaired in both conventional- and UHDR-irradiated female nude (NUto 10 Gy . These dIn the context of cancer involving (brain) tumors, reduced neutrophil levels are likely also beneficial for tumor control, as neutrophils were shown to promote tumor resistance to radiotherapy, while a lower neutrophil count following chemoradiotherapy was associated with higher rates of local tumor control, metastasis-free survival, and overall survival ,20,21. C−6 s per pulse, which is similar to several past studies with electrons [Recent studies of UHDR irradiation on cognitive performance have used a variety of irradiation delivery systems. They include electron ,11,27, Xlectrons ,9,27; hoIn the current study, there was a trend toward increased measures of anxiety in the elevated zero maze. Consistent with this trend, when activity levels in the center of the open field were assessed by beam brakes in neonatally irradiated rats, they were lower in conventional- and UHDR-irradiated rats that received 8 Gy and conventional-irradiated rats that received 5 Gy and they seemed lower in 5 Gy UHDR-irradiated rats as well, although it did not reach significance . These dWe did not see reduced cognitive injury following UHDR, as compared to conventional cranial irradiation. Together with other studies, these data suggest that the protective cognitive effects of UHDR irradiation might be very dose-dependent and seen at lower and higher doses than those used in the current study. In addition, there is likely a dependence on cognitive tests used to compare conventional to UHDR irradiation. Protective effects of UHDR irradiation for novel object recognition memory were seen at doses of 8, 10, or 12 Gy in female mice but not when the dose was 14 Gy ,12,29. PInfiltration of immune cells into the brain might play a role in cognitive injury. For example, in mice with tauopathy and in Alzheimer’s disease (AD) brains, there is microglia-mediated T-cell infiltration-induced tauopathy, a marker of AD neuropathology . The numWhile cognitive injury was comparable in conventional and UHDR mice, the percentage of CD45+ CD11b+ Ly6+ cells in the hippocampus cells in the hippocampus was altered in conventional- but not UHDR-irradiated mice. As the percentage of CD45+ CD11b+ Ly6+ cells correlated negatively with the percentage of entries in the novel arm of the spatial Y maze, a cognitive measure not affected by conventional irradiation, and CD11+ Ly6G+ cells were shown to inhibit tumor growth , these dMice and irradiation. Two-month-old female and male C57BL/6J mice  purchased from JAX labs received whole brain conventional or UHDR irradiation. In order to not limit the response to whole brain conventional or UHDR irradiation to one sex, both sexes were included in the current study. One UHDR-irradiated female mouse died, so there were five mice in that group. UHDR dose mice were irradiated with 9 MeV electrons with a target dose of 18 Gy delivered by the Linac-based/modified beam control. The delivered UHDR dose was verified by GAF chromatic film after irradiation. The conventional dose rate was delivered to match the UHDR. Twenty-four hours is typically allowed between UHDR and conventional irradiations to allow the film to stabilize, however, scheduling constraints prevented this stabilization time between irradiations (in these mice). Early estimates from the film (within 1 h of irradiation) indicated a delivered dose of 18 Gy UHDR and so this dose was delivered to the conventional-irradiated group. Conventional mice were given 18 Gy  to match the UHDR mice. After 24 h passed, the films were reanalyzed and it was found that the early estimates were high, only 17 Gy was delivered in the UHDR group  were provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committees at Dartmouth and OHSU and were in compliance with all federal regulations. Ten weeks following radiation or sham irradiation, the mice were euthanized by cervical dislocation, and their hippocampi were analyzed for alterations in the numbers of immune cells by flow cytometry.Spontaneous alternation in the Y maze. Spontaneous alterations were assessed in a Y-shaped maze with raised sides  and made of non-reflective opaque gray plastic (37.98 cm length)  as described in [ribed in . At the Novel object recognition. In this test, the mice were put in the open field containing two identical objects  for a 15 min trial. The objects were placed 10 cm apart and 15 cm from the adjacent walls of the arena. The following day, one object was replaced with a novel one (a green wooden block in the shape of a triangular prism). Mice were again allowed to explore for 15 min. Objects were affixed to the floor of the arena using masking tape. The arenas and objects were cleaned with 0.5% acetic acid between trials. Physical interaction with the object in the form of sniffing within a 2 cm proximity was coded as object exploration by hand scoring videos acquired with Noldus Ethovision software . In addition to the percentage of time spent exploring the novel and familiar objects, a discrimination index was calculated. The time spent exploring the familiar object was subtracted from the time exploring the novel object, and the resulting number was divided by the total time spent exploring both objects. Elevated zero maze. Measures of anxiety were assessed in the elevated zero maze as described in [ribed in . The eleLight–dark test. In the light–dark test, mice were placed in an open-field enclosure (40.64 × 40.64 cm) containing black plastic inserts covering the sides and the top 50% of the open field , as described in [ribed in . A singlSpatial Y maze. The spatial Y maze test had raised sides and was made of non-reflective opaque gray plastic (30 cm × 6 cm × 15 cm) , as described in [ribed in . On day Fear learning and cued fear memory. Fear conditioning was assessed over the course of 2 consecutive days using a Med Associates mouse fear conditioning system  and Med Associates VideoFreeze automated scoring system, as described in [ribed in . Mice weGrip Strength. Grip strength was assessed using a Harvard Apparatus  grip strength meter for mice. The grip strength meter allows the study of neuromuscular functions in rodents by determining the maximum force displayed by an animal. The grip strength meter was positioned horizontally and the mice were held by the tail and lowered toward the apparatus. The mice were allowed to grasp the metal grid and were then pulled backward in the horizontal plane. The force applied to the grid just before the mouse lost grip was recorded as the peak tension. We performed 3 consecutive measurements at one-minute intervals, as described in [ribed in  . The hippocampus of one hemibrain was collected in 300 μL of RPMI. Flow cytometry was performed using hippocampi from one hemibrain as described in [ribed in , with thFlow cytometry. Spectral compensation was achieved by using compensation beads  conjugated to the above-mentioned antibodies in 100 μL of FACS buffer and following a vendor-recommended protocol. Following vortexing the comp beads, one drop of each bottle of com beads was added and the tubes were incubated for 20 min at room temperature. After adding 3 mL of FACS buffer, the tubes were centrifugated at 400 rcf for 5 min. The supernatants were aspirated and the pellets were resuspended in 500 μL of FACS buffer.Data for flow cytometry were acquired on a BD FAC Symphony A5 (BD Immunocytometry Systems) equipped with 405 nm , 488 nm , and 628 nm  excitation lasers. Data were collected and analyzed using BD FACS Diva Software v8.0.2 (BD Biosciences) and FlowJo software v10.1 . As female and male samples were analyzed on separate days, the percentage of the subpopulation of CD45+ Ly6G+ cells was normalized to the sex-matched sham-irradiated samples analyzed on the same day. Cells were first gated on FSC and SSC in order to eliminate debris. They were then gated on FSC-W and SSC-W in order to reduce the analysis of doublets. Cells that were negative for the viability marker, Ghost Red, were gated on. After this, CD45 cells were gated on and Ly5G, CD11b, and CD206 were all analyzed as a subset of CD45.Data analyses. Data are expressed as mean ± SEM. Graphs were generated using GraphPad software v.8.2.0 . Data were analyzed using GraphPad and SPSS software . The data were analyzed using ANOVAs with exposure conditions as between-group factors, followed up by post hoc tests when appropriate. Performance over multiple trials was analyzed by repeated-measures ANOVA. For assessing relationships between flow cytometry and behavioral measures, Spearman correlational analyses were used.


The OP decreased by high-speed sintering in the DL for Cercon xt ML and in all the layers for Lava Esthetic (p < 0.05). The decrease in translucency after high-speed sintering was attributed to a decrease in the yttria-rich t’-phase with low tetragonality, along with an increase in the yttria-lean t-phase with high tetragonality.As dental 5 mol% yttria-stabilized (5Y-) zirconia demand high esthetics, it is necessary to clarify how the optical properties are affected by high-speed sintering, which is not yet fully understood. Our study aimed to investigate the effect of high-speed sintering on the translucency and opalescence parameters , as well as their related microstructure and phase distribution, using two types of multilayered 5Y-zirconia. Multilayered 5Y-zirconia  were cut layer-by-layer, followed by conventional and high-speed sintering. The TP and OP values were subsequently obtained using a spectrophotometer, and field emission scanning electron microscopy images were used to analyze the average grain size. The phase fractions were analyzed using X-ray diffraction. Regardless of the zirconia type, the TP was slightly lowered by high-speed sintering in all the layers except the dentin layer (DL) for Lava Esthetic ( Natural human teeth possess various optical properties, such as translucency, opalescence, and fluorescence ,2,3,4,5.Apart from being translucent, natural teeth also exhibit opalescent properties . Owing t2-Y2O3 system [Traditionally, the manufacture of zirconia prostheses involves a lengthy sintering process . However3 system . Based o3 system . Consequ3 system . The hig3 system ,23, and Recently, advances have been made in the production of zirconia with improved translucency and a multilayered structure, as well as the investigations about the multilayered ceramics . The mul3 (±0.008).The zirconia  used in A spectrophotometer  was used to analyze the optical properties based on the ISO/TR 28642 [00) was calculated with the values obtained from reflectance measurements against white and black backgrounds using the following equation [To obtain TP of the specimen, the CIEDE2000 color difference CIL, KC, and KH are parametric factors used to compensate for the mismatch in the experimental conditions; they were fixed at 1 in the present study [L, SC, and SH correspond to the weighting functions for lightness, chroma, and hue, respectively. RT represents the rotation function, which is utilized to adjust for the interaction between the differences in chroma and hue in the blue region. To evaluate the ΔE00 value, perceptibility thresholds of 50:50% (ΔE00 = 1.30 and 1.25 for the fitted S-shape curves and Takagi-Sugeno-Kang (TSK) Fuzzy Approximation, respectively) and acceptability thresholds of 50:50%  were used [Knt study ,31. SL, OP was obtained using the following equation ,13OP = T − a*R) and (b*T − b*R), respectively.Here, subscript T represents the transmitted light and subscript R represents the reflected light measured against a black background . Δa* andTo analyze the microstructures of the specimens (n = 2/group), FE-SEM examination  was performed. The accelerating voltage was set to 10 kV. Prior to the analysis, a platinum sputter coating was applied to the specimens for 90 s. The obtained FE-SEM images were used to analyze the crystal size of each layer based on the standard ASTM E112. The linear intercept method  was empl2/nmc, Fm3m, and P21/c, respectively [2O3 (mol%) content based on the lattice parameters  of the tetragonal phase, the following equations were used [For high-resolution XRD analysis, Ni-filtered CuKα radiation was used  in the range of 25–90 degrees (2θ). The step size used for the analysis was 0.013 degrees (2θ), and the tube current and voltage were 30 mA and 40 kV, respectively. XRD patterns were subjected to Rietveld analysis using Topas Academic software V 7.21 . The fractions of the tetragonal, cubic, and monoclinic phases were determined. The space groups of the tetragonal, cubic, and monoclinic phases were P4ectively . The staectively , Lamas aectively , and Smiectively . The refere used ,27:(4)YOStatistical analysis was conducted using statistical software SPSS 25.0  and a significance level of 0.05 was set. The data’s normality was assessed using the Shapiro–Wilk test. To analyze the effects of zirconia type, sintering speed, and each layer of zirconia on TP, OP, Δa*, and Δb*, a three-way analysis of variance (ANOVA) followed by a post hoc Tukey’s HSD test was conducted. Because the grain size did not meet the assumption of normality, non-parametric methods  were used. For grain size comparisons between the layers, the Mann–Whitney U test was employed.p < 0.001) and layer (p < 0.001) had a significant effect on the TP. An interaction was observed between the type of zirconia used and the layer (p < 0.001).The results of the three-way ANOVA  suggestep < 0.05). Cercon x t ML showed a slight increase in TP from the enamel layer (EL) to the DL at both sintering speeds (p < 0.05). Conventionally sintered Lava Esthetic showed a slight decrease in the TP from EL to DL (p < 0.05). The TP values were not significantly different among the layers for the high-speed-sintered Lava Esthetic. When the sintering speed was the same, the TP values were not significantly different between the two zirconia samples .High-speed sintering  resultedp = 0.002), sintering speed (p < 0.001), and layer (p < 0.001) had a significant effect on the OP. An interaction was observed between the type of zirconia used and sintering speed (p = 0.004), as well as between the sintering speed and layer (p = 0.018).The findings derived from the three-way ANOVA  suggestep < 0.05). The OP values were not significantly different between the two zirconia when conventionally sintered (p < 0.05). However, with high-speed sintering, Lava Esthetic demonstrated lower OP values than Cercon xt ML in the EL and transition2 (T2) layers (p < 0.05).High-speed sintering decreased the OP only in the DL for Cercon xt ML and in all the layers for Lava Esthetic . Both Cep < 0.05). Both Δa* and Δb* increased from EL to DL, irrespective of the type of zirconia and sintering speed. The increase in Δa* was minimal, whereas the increase in Δb* was more noticeable.High-speed sintering decreased Δa* in all the layers for both zirconia types; however, Δb* decreased only in the DL for Cercon xt ML and in all the layers for Lava Esthetic (p < 0.05); however, the difference was minimal. Neither type of zirconia exhibited significant differences in the crystal size between the EL and DL. The crystal size of all the specimens was approximately 1–1.2 μm.SEM observations revealed that both types of zirconia exhibited equiaxed crystal structures with a mixture of larger and smaller crystals . No appat’) and tetragonal (t) phase, at both sintering speeds. Both types of zirconia exhibited a slight reduction in the t’-phase and a slight increase in the t-phase in both the EL and DL after high-speed sintering. The difference in the phase content between the EL and DL was small. Irrespective of the type of zirconia used and the sintering speed, the axial ratio  of the t’-phase was ~1.005, while the axial ratio of the t-phase was ~1.015. Additionally, the t’-phase exhibited a higher yttria content compared to that of the t-phase.An analysis was performed on the XRD patterns  of the sp < 0.05). For Katana STML (5Y), for which the manufacturer provides a high-speed sintering schedule, the TP value did not change with high-speed sintering [00 = 1.30) [This study was conducted to investigate the differences in the optical properties, microstructure, and phase fraction between high-speed sintered and conventionally sintered multilayered 5Y-zirconia. The experimental results provided partial evidence against the null hypothesis, suggesting that high-speed sintering has an effect on the optical properties, microstructure, and phase fraction of the zirconia used. The TP value slightly decreased in all the layers (except the DL of Lava Esthetic) by high-speed sintering, regardless of the zirconia type  . Therefo = 1.30) . However = 1.30) .p < 0.05) after conventional sintering, while no significant interlayer TP difference was observed following high-speed sintering. In contrast, Cercon xt ML showed a slight increase in TP from the EL to DL, regardless of the sintering speed (p < 0.05). Therefore, the changes in translucency from the EL to DL varied according to the type of zirconia and sintering speed; however, the TP changes from the EL to DL were lower than the perceptibility thresholds of CIEDE2000 (ΔE00 = 1.30) [Lava Esthetic showed a slight decrease in TP from the EL to DL ( = 1.30) . The mul = 1.30) ,37, whic = 1.30) ,16.00 = 1.30) [Irrespective of the sintering speed, the difference in the TP values between Lava Esthetic and Cercon xt ML was below the perceptibility thresholds by CIEDE2000 ΔE = 1.30) . The TP  = 1.30) ,38. For  = 1.30) . The TP .30 . The = 1.30) . In the  = 1.30) .p < 0.05). A slight decrease in the OP after high-speed sintering was also reported for 4Y- and 6Y-zirconia, and their OP values were similar to those of the 5Y-zirconia in this study [Natural human teeth exhibit opalescent properties in addition to their translucent properties . Owing tis study . The OP is study . Δa* decis study .p < 0.05). This trend was also reported for 4Y- and 6Y-zirconia [The two types of zirconia used in this study were multilayered zirconia with a color gradient. The OP values increased from the EL to DL, regardless of the sintering speed, for both types of zirconia  of the t’-phase was approximately 1.005, while the axial ratio of the t-phase was approximately 1.015. Therefore, the t-phase exhibited higher tetragonality than the t’-phase. These findings are consistent with those reported in the literature [t-phase has tetragonality, which causes optical birefringence and decreases translucency. The metastable t’-phase has higher yttria content than does the t-phase [t’-phase, resulting in higher translucency compared to that in the t-phase [t’-phase with low tetragonality, along with an increase in the yttria-lean t-phase with high tetragonality. Similarly, in a study where Katana STML (5Y) was high-speed-sintered at 1560 °C with a holding time of 6.9 min, a decrease in the cubic phase was observed along with an increase in the tetragonal phase with high tetragonality [t-phase decreased while the t’-phase increased, contradicting the findings of our study [t’-phase, the translucency decreased owing to the increased number of residual pores caused by high-speed sintering, as well as the decrease in flexural strength [Based on the Rietveld analysis of the XRD data, both types of zirconia showed a slight decrease in the metastable terature ,27. The  t-phase ,27, as o t-phase . Based ogonality . In a stThis study had certain limitations that should be acknowledged. First, the study used only two types of zirconia, which may not fully represent the entire range of available zirconia materials. Consequently, the findings of this study may not be applicable to other types of zirconia. Additionally, variations in the high-speed sintering conditions were limited. There was also the lack of aging for the long-term evaluation of the investigated variables. Consequently, the generalizability of the study’s conclusions may be restricted to the specific conditions investigated. Thus, different results can be obtained using zirconia from different manufacturers or by altering the protocol for high-speed sintering. To obtain more conclusive results, further studies are needed. t’-phase, accompanied by a slight increase in the yttria-lean t-phase.Regardless of the type of zirconia, high-speed sintering resulted in a slight decrease in the TP in all layers (except the DL for Lava Esthetic). After high-speed sintering, the OP decreased only in the DL for Cercon xt ML, whereas for Lava Esthetic, it decreased in all the layers. These changes in the OP corresponded to the changes in Δb*. The minor reduction in translucency owing to high-speed sintering was attributed to a slight decrease in the yttria-rich


Introduction: Intracerebral microglia play a vital role in mediating central immune response, neuronal repair and synaptic pruning, but its precise role and mechanism in fast action of antidepressants have remained unknown. In this study, we identified that the microglia contributed to the rapid action of antidepressants ketamine and YL-0919.Methods: The depletion of microglia was achieved with the diet containing the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 in mice. The tail suspension test (TST), forced swimming test (FST) and novelty suppressed feeding test (NSFT) were employed to evaluate the rapid acting antidepressant behavior of ketamine and YL-0919 in the microglia depletion model. The number of microglia in the prefrontal cortex (PFC) was assayed by the immunofluorescence staining. The expressions of synaptic proteins  and brain-derived neurotrophic factor (BDNF) in the PFC were tested by Western blot.Results: The immobility duration in FST and the latency to feed in NSFT were shortened 24 h after an intraperitoneal (i.p.) injection of ketamine (10 mg/kg). The microglial depletion of PLX3397 blocked the rapid antidepressant-like effect of ketamine in mice. In addition, the immobility time in TST and FST as well as latency to feed in NSFT were reduced 24 h after the intragastric (i.g.) administration of YL-0919 , and the rapid antidepressant effect of YL-0919 was also blocked by the microglial depletion using PLX5622. About 92% of microglia in the prefrontal cortex was depleted in PLX5622 diet-fed mice, while both ketamine and YL-0919 promoted proliferation on the remaining microglia. YL-0919 significantly increased the protein expressions of synapsin-1, PSD-95, GluA1 and BDNF in the PFC, all of which could be blocked by PLX5622.Conclusion: These results suggested the microglia underlying the rapid antidepressant-like effect of ketamine and YL-0919, and microglia would likely constitute in the rapid enhancing impact of synaptic plasticity in the prefrontal cortex by YL-0919. Depression, as an affective disorder, is characterized mainly by persistent and recurrent dysthymia, and has become a serious global public health challenge. The pathogenesis of depression is still unclear. The clinical antidepressants are mainly based on “monoamine strategy,” such as selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs). Most of first-line antidepressants have defects such as delayed onset of action, limited efficacy (50%–70%), cognition impairment, sexual dysfunction and possible suicidal tendency . The newAs the resident immune cell in the central nervous system (CNS), microglia was related to the pathogenesis of depression . The levs-ketamine), and extensive clinical evidences demonstrated that a single subanesthetic dose of ketamine produced antidepressant efficacy lasting for 1 week (R)-ketamine on chronic social defeat stress (CSDS) susceptible mice, suggesting that microglia might be involved in the antidepressant effect of (R)-ketamine . The effect of microglial depletion on the rapid action of ketamine and YL-0919 was identified by CSF1R inhibitor PLX5622 in mice. The microglial depletion ratio of PLX5622 and the effect of ketamine/YL-0919 on the number of microglia were assessed by immunofluorescence in the PFC. At last, the impact of microglial depletion on the YL-0919-induced upregulated synaptic proteins and BDNF were observed to demonstrate a new antidepressant mechanism of YL-0919.Male adult C57BL/6J mice, aged 8 weeks (21–23 g) were purchased from SPF (Beijing) Biotechnology. The mice were housed in groups of 5 per cage at constant temperature (22°C ± 2°C), in a humid environment (55% ± 10%) and exposed to a light/dark cycle of 12 h for 7d of acclimatization. The mice were provided with food and water freely. All procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the institutional committee on animal care and use.YL-0919 was provided by the Institute of Pharmacology and Toxicology (with purity>99.9%). Ketamine was purchased from Fort Dodge Animal Health . Ketamine and YL-0919 was dissolved in distilled water and administered respectively at a volume of 10 mL/kg. The depletion of microglia was achieved by the CSF1R inhibitor PLX5622 for 19 days . On the sixteenth day of PLX5622 or vehicle chow (AIN-76A) treatments, ketamine  was administered intraperitoneally 24 h prior to behavioral tests. Mice were divided into 4 groups, and each group contained 9-10 mice.On the tenth day of PLX5622 or vehicle chow (AIN-76A) treatments, YL-0919  was administered twice (8:00 a.m. and 8:00 p.m.) for 5 consecutive days, and was then treated once (8:00 p.m.) during the testing period. Behavioral tests were performed separately 24 h after the administration of YL-0919. Mice were divided into 4 groups, and each group contained 11-12 mice. The procedures of drug treatments and tests were demonstrated in The LA was performed following previously described procedures . The micThe TST was performed following previously described procedures . The MedThe FST was performed following previously described procedures . The micThe NSF was performed following previously described procedures . The micThe mice were anesthetized with 1% pentobarbital sodium and perfused with 4% paraformaldehyde (PFA). Brains were extracted and fixed in 4% PFA. The tissues were stored in different sucrose solutions for gradient dehydration and then sectioned with a cryostat at 30 μm. The sections were washed with PBS buffer (pH = 7.4) for 3 times and blocked with PBS containing 0.3% Triton-X and 10% normal goat serum at room temperature for 2 h. Sections were then incubated with primary antibody of Iba1  overnight at 4°C, Following washes in PBS, the tissues were incubation with biotinylated goat anti-rabbit IgG secondary antibody  for 2 h at room temperature in the dark. After washing, the sections were mounted and cover-slipped with anti-fluorescence quenching agent containing DAPI on slides. Immunofluorescence imaging was examined using confocal microscopy (Nikon A1). ImageJ was measured to analyze the number of microglia.The mice were decapitated for sacrifice, and the prefrontal cortex was dissected and stored at −80°C. Cortical tissues were homogenized, and centrifuged at 12,000×g for 10 min. The supernatant was recovered, and protein concentration was evaluated using BCA method. Samples containing 30 μg of proteins were loaded onto the SDS polyacrylamide gel and transferred to PVDF membranes. The membrane was blocked with 5% skim milk, and incubated separately with primary antibodies of Iba1, synapsin-1 , GluA1 , PSD-95 , BDNF , sigma-1  or β-actin  overnight at 4°C. The membrane was washed 3 times with TBST, and incubated in fluoresce-labeled secondary antibodies for 2 h at room temperature in the dark. Images was visualized using an Odyssey two-color infrared imaging system.p < 0.05 was considered statistically significant.The statistical data are represented as Means ± SEM. In the behavior tests, experiments were performed blind. Results were analyzed using Student’s t-test or the two-way ANOVA followed by Tukey’s multiple comparisons test. The p > 0.05). Compared with the control group, the immobility durations in FST and NSFT were significantly decreased by ketamine  injection, indicating a rapid acting antidepressant effect of ketamine . In addition, the immobility durations of mice in FST and TST were significantly reduced by the administration of YL-0919  . The latency to feed was also reduced by YL-0919 (administered for 9 consecutive days) . YL-0919 preformed a rapid acting antidepressant effect, which was consistent with the onset time of action from previous studies in our lab  = 2.996, p = 0.0246; p < 0.05]. Compared to YL-0919 group, microglial depletion by PLX5622 also significantly blocked the fast antidepressant action of YL-0919 for increased immobility durations of TST, FST and latency to feed . However, PLX5622 itself had no impact on the above behaviors. These results suggested that microglia played an essential role in fast antidepressant action of ketamine and YL-0919.To investigate whether microglia contribute to fast action of ketamine and YL-0919, depletion of microglia in mice was achieved with the diet containing PLX5622 . There w our lab . Interesp > 0.05). After PLX5622 diet, the density of microglia in the PFC was reduced by 92% , and the expression of Iba1 protein in the PFC was significantly decreased . However, both ketamine and YL-0919 promoted the proliferation of the remaining microglia in the PFC of mice treated with PLX5622 .The effect of PLX5622 on microglial density and the regulation of ketamine and YL-0919 on the number of microglia were validated by immunofluorescence staining in the PFC . Comparep = 0.0349; t = 3.949, p = 0.0042; t = 2.398, p = 0.0433; t = 2.968, p = 0.0179; t = 2.839, p = 0.0218; p < 0.05). Compared with the YL-0919-treated group, the upregulation of GluA1, PSD-95, Synapsin-1, and BDNF by YL-0919 were significantly blocked by PLX5622 . Mounting studies suggested a crucial role of BDNF in rapid antidepressant action , which was blocked by PLX5622 . These results suggested that YL-0919 promoted the expression of synaptic proteins and BDNF with the rapid antidepressant action simultaneously, and microglia were significantly involved in the increased expression of synaptic plasticity, BDNF and sigma-1 receptors by YL-0919 treatment.To investigate the possible microglial mechanism of the rapid antidepressant action of YL-0919, the expression of synaptic proteins , BDNF, and sigma-1 receptor in the PFC were assayed. YL-0919  significantly increased the expression of synaptic proteins  and BDNF in the PFC  produced a rapid acting antidepressant effect in chronic unpredictable stressed (CUS) rodents within 3–5 days . In addiR)-ketamine in CSDS susceptible mice (R)-ketamine (Microglia are among the most potent modulators of neuronal activity and CNS homeostasis . Adjustmble mice , suggestble mice . TGF-β1 ketamine .Previous studies showed that YL-0919 treatment for 5 days significantly activated the BDNF-mTOR pathway and increased the synthesis of synaptic proteins and BDNF underlying enhanced dendritic complexity in the PFC . The actMicroglia are the only type of cells that express CSF-1R in the CNS, and CSF-1R inhibitor PLX5622 can deplete specifically cerebral microglia . In thisIn addition, our recent studies demonstrated that YL-0919 was a high-affinity agonist of the sigma-1 receptor . ActivatIn conclusion, the current data revealed that microglia contributed to the rapid antidepressant of ketamine and YL-0919, and microglia participated in the upregulation of synaptic plasticity by YL-0919. Therefore, it is likely that microglial regulation would be a new direction for fast onset of anti-depression.


Six treatments with mild water-filtered infrared-A WBH over a period of three weeks with at least one day in between treatments were applied. On average, the maximum temperature was 38.7 °C for a duration of approximately 15 min. The control group received exactly the same treatment except that an insulating foil between the patient and the hyperthermia device blocked most of the radiation. Primary outcome was pain intensity measured by the Brief Pain Inventory at week 4. Secondary outcomes included blood cytokine levels and FMS-related core symptoms and quality of life. Pain intensity at week 4 was significantly different between the groups in favor of WBH (p = 0.015). A statistically significant pain reduction in favor of WBH was also found at week 30 (p = 0.002). Mild water-filtered infrared-A WBH effectively reduced pain intensity at the end of treatment and follow-up.The challenging treatment situation of patients with fibromyalgia syndrome (FMS) requires additional therapy options. The effects of water-filtered infrared-A whole-body hyperthermia (WBH) versus sham hyperthermia on pain intensity were investigated in an outpatient setting within a two-armed randomized sham-controlled trial.  Fibromyalgia syndrome (FMS) is a chronic disorder characterized by chronic widespread pain, physical and/or mental fatigue, and nonrestorative sleep as core symptoms. It is often accompanied by various somatic and psychological symptoms such as headache, bowel problems, morning stiffness, anxiety, and depression. The worldwide prevalence varies between 0.2 and 6.6% . Women wThe treatment situation of FMS is often perceived as insufficient ,10,11,12WBH works by increasing body-core temperature to create an artificial fever-like state . AdverseThe main objective of this randomized sham-controlled trial was to investigate the effect of mild wIRA-WBH on pain intensity in patients with FMS in an outpatient setting.https://clinicaltrials.gov/ct2/show/NCT05135936 (accessed on 13 February 2023), and performed according to the declaration of Helsinki applying good clinical practice standards.This prospective, monocentric, randomized, sham-controlled, single-blinded, 2-armed, parallel group trial was conducted from November 2020 to December 2021 at the Sozialstiftung Bamberg, Bamberg, Germany. The study was approved by the Ethics Committee of the Bayerische Landesärztekammer , registered on clinicaltrials.gov  aged between 18 and 70 years with a medically confirmed diagnosis of FMS additionally reviewed by a physician according to ACR 2016 criteria during the screening visit and an average pain intensity of ≥4.0 were eligible. The latter was recorded via a pain diary, measuring the pain level four times a day 14 days prior to the eligibility assessment. Main exclusion criteria were severe somatic and psychiatric comorbidities, other chronic pain syndromes, the intake of opioids, cannabis, and immunosuppressive drugs, contraindications for hyperthermia such as body temperature >37.5 °C, heart failure, previous experience with WBH, or the participation in other clinical intervention studies see .After being assessed as eligible by the study doctor, patients were randomly assigned to either the intervention or the control group by the study coordinator (SC). For that purpose, the study coordinator prepared opaque sealed envelopes containing either “A” (for intervention) or “B” (for control) in a 1:1 ratio with a block size of 10. The SC asked the patients to select one envelope, open it and to say out loud the containing letter, which was then protocolled. The meaning of the letters and group allocation was blinded to the patients, study doctor, and those being involved in the assessment of outcomes  but not to the SC and therapists.Participants assigned to the intervention group received a total of six sessions with mild wIRA-WBH of 60 min over a period of three weeks with at least one day between each intervention session.The IRATHERM1000 hyperthermia device was used for this purpose  and can be adjusted in 5% intervals by a control panel (2) during the heating-up phase. When reaching the target temperature, irradiance was decreased to 40% (560 W/m2) to maintain body-core temperature until the end of the 60-min treatment period (plateau phase). This led to a further small increase in temperature in most of the subjects. Deviations from the planned irradiance were made according to the needs of the participants. While being treated subjects laid undressed on the device being covered by a white sheet and a reflection foil.The device stands out for its open design and lying comfort. It has six halogen radiators arranged longitudinal with three on each side including water. The resulting spectrum is called water-filtered infrared-A (wIRA), with typical absorption lines of water and free of infrared B and C. For wIRA, the relationship between irradiation of the skin and deeper lesions is much more favorable compared to conventional technical IR-devices. Maximum irradiance is 1400 W/mol panel B for eac2) were recorded continuously during the entire session and could be viewed at any time by the therapist, but not by the participant. After treatment, participants rested for about 30 min (resting-phase). Thus, one complete intervention was approximately 1.5 to 2 h. Subjects were permanently supervised and a physician was always on call. A discontinuation criterion of the treatment was a body-core temperature of more than 40 °C.Rectal and axillar temperature, pulse and oxygen saturation . Clinical characteristics were captured at baseline (week 0), one week after the end of treatment (week 4/postintervention), and two and six months after the end of the treatment .The primary outcome was pain intensity measured by the Brief Pain Inventory (BPI) ,38 at wen = 28 patients . Sensory and affective pain were measured using a subscale of the Short-Form McGill Pain Questionnaire (SF-MPQ) [Pain impairment was measured by the BPI ,38. FMS-(SF-MPQ) . Somatic(SF-MPQ) . It cont(SF-MPQ) . All queg, 10 min, 4 °C) and stored at −80 °C until analyses. Plasma cytokine levels were determined by high-sensitive enzyme-linked immunosorbent assays (ELISA) with detection limits of 0.022 pg/mL for TNF-α, 0.031 pg/mL for IL-6, 0.130 pg/mL for IL-8, and 0.090 pg/mL for IL-10. Serum concentrations of CRP, ESR and WBC differential were analyzed by the local clinical laboratory.Blood samples were collected at baseline (T0), immediately after the last treatment session (T1) and at the following week  to determine systemic concentrations of key pro- and anti-inflammatory cytokines . Average daily interval of T1 and T2 blood collection was 4.16 ± 1.08 days. At these timepoints, also a routine laboratory testing with C-reactive protein (CRP), erythrocyte cell sedimentation rate (ESR) and white blood cell (WBC) differential was performed. Venous blood was collected in tubes containing EDTA  and plasma was separated by centrifugation  was reported as an effect-size estimator. A partial eta square of 0.01, 0.06, and 0.14 corresponds to a small, medium, and large effect, respectively. All results are reported as mean ± standard deviation (M ± SD). Analyses were performed using the Statistical Package for Social Sciences software .Intention-to-treat analyses were performed. Missing values were imputed 50 times using the Markov–Chain–Montecarlo-procedure. The primary outcome was evaluated using an analysis of covariance (ANCOVA) with baseline scores as covariate to control for group differences in pain intensity at baseline and with group as between-subject factor. Blood parameters were evaluated using multivariate repeated-measures analysis of variance (rmANOVA). Post hoc d = 0.60) with clinical relevance was assumed based on prior results by Brockow et al. [A moderate effect size  and analyzed  and were medically assessed for eligibility at the clinic. Finally, 41 subjects (93%) fulfilled inclusion and exclusion criteria and were randomized .The intervention group showed an average reduction in pain intensity from baseline of 1.7 points , while pain intensity in the control group was reduced by 0.5 points . In addition, a clinically relevant reduction in pain of 30% from baseline was found in 10 patients of the intervention group and in 4 patients of the control group  with an average reduction of pain intensity of 0.9 points . Pain intensity in the control group was increased by 0.5 points . Pain impairment measured at week 30 was statistically significantly different between the groups in favor of mild WBH . Mental health index score as measured with the SF-36 at week 4 showed statistically significant differences between the groups in favor of mild WBH . Secondary outcomes are presented in p-values.A statistically significant pain reduction in favor of the intervention group was found at week 30  and neutrophil counts .Analyses including all blood parameters as outcome variables revealed a statistically significant time × group interaction for lymphocyte  at T1  with a greater increase in lymphocyte count by mild WBH (p = 0.033) at T2  with higher scores in the mild WBH group (p ≤ 0.001 (B = −0.905 ± 0.257) and in the timeframe T0 to T2 with p = 0.017 (B = −0.984 ± 0.402).Post hoc mild WBH A. NeutroBH group B. Multilp = 0.027, B = −0.285 ± 0.126) on pain intensity, detecting that an increase in IL-6 concentration results in a statistically significantly less pain intensity (timeframe T0 – T1).Analyses showed no significant time × group interaction for cytokines. However, multilevel analyses revealed a negative effect of IL-6  and neutrophils  on IL-6 regarding the time course .A fixed-effects model showed statistically significant positive effects of lymphocytes  and rise in body-core temperature . There is a significant negative correlation between lymphocyte cell count (T0) and the maximum temperature reached at the first session, r = −0.510, p = 0.026, i.e., the lower the lymphocyte cell count (T0) was, the higher the body-core temperature was afterwards.A total of 18 subjects in each group, corresponding to 86% for intervention, resp. 90% for control, received the full treatment series of six interventions. Mean duration of heating-up phase in the intervention group was 45.30 ± 6.59 min (range: 35.17–57.17 min) and average plateau phase was 14.69 ± 6.64 min (range: 3.00–24.83 min). Subjects of the intervention group had a mean body-core of 38.7 ± 0.2 °C at the end of the treatment, resulting in an average rise of 1.5 ± 0.3 °C. The control group showed a highest average body-core temperature of 37.5 ± 0.3 °C, resulting in a mean rise of 0.3 ± 0.2 °C. As expected, the intervention group had a significantly higher average maximum body-core temperature . More temperature data, pulse rate, and oxygen saturation can be found in In addition, the intervention group showed a higher difference in baseline body-core temperature between the first and the sixth treatment, indicating a higher reduction in body-core temperature in mild WBH  were convinced to be part of the active condition, while 16 of 20 control patients (80%) were assuming this.In the intervention group one patient missed the second treatment due to dizziness and redness after the first session of mild WBH. Another patient experienced severe dizziness and circulatory problems during the first treatment. Therefore, she received an infusion to stabilize her general condition. Side effects did not lead to a discontinuation of the treatment series in any patient of the intervention group. One patient of the control group discontinued the treatment series after the fourth session due to fatigue. A detailed presentation of side effects during the interventions, based on the reports of the therapists, is provided in First, pain intensity 1 week and 6 months after the end of the treatment was significantly lower in the wIRA-WBH group compared to the control after the intervention series. A midterm sustained pain reduction can be achieved by wIRA-WBH. In addition, exploratory analyses revealed a reduction in pain impairment in the intervention group at week 30 and better mental health at week 4. Second, no severe adverse events were observed in either the intervention or the control group which indicates safety of mild wIRA-WBH, and tolerability was high. There were no drop outs due to side effects. Third, clinical results were supported by immunological data which showed a significantly higher increase in immune cell counts in the wIRA-WBH group. Higher blood lymphocyte numbers and IL-6 levels appeared with lower pain intensity.The observed effects on pain intensity are in line with the previous controlled trials of wIRA-WBH in FMS ,19,22, tThe generally mild side effects are in line with the previously reported studies on mild wIRA-WBH in FMS . Body-cThe mental health summary score of SF-36 questionnaire was higher in the intervention group at week 4. This is interesting as the physical part of SF-36 showed no difference between both groups. In the previous controlled trials on WBH in FMS, Romeyke and Stummer  focused Though FMS is considered a chronic syndrome rather than an inflammatory disease, it exhibits distinct systemic immunological patterns, including changes in neutrophil and lymphocyte counts and elevated levels of IL-6, IL-8, and TNF ,52. ImmuThus, pain relief could have been achieved by thermotherapy-induced immune stimulation. In addition, immunological processes are supported by the higher reduction in body-core temperature in mild WBH, and may also indicate physiological adaptation as part of the strong stimulus of the therapy. Another interesting observation is the inverse correlation of lymphocyte cell number with the maximum body-core temperature reached at first session. Further studies should examine the coherence between immune system and nervous system, and the presence of chronic pain.To our knowledge, the study is the first to investigate the effects of mild wIRA-WBH in patients with FMS in an outpatient setting. The main strength of the present trial was the establishment of an adequate sham condition with high credibility in the framework of a randomized controlled setting. Further, a priori sample size calculation ensured that the study was sufficiently powered to detect the group differences. The immunological data allow a combination of subjective and objective parameters and thus further supports the validity of the results.Even if the control condition of the present trial is considered a main strength, it should be noted that it was no traditional placebo treatment, since a small amount of the postulated key factor (heat) was applied as well. However, this was considered the only way to construct a credible control condition in heat applications and effects should be limited. Besides the monocentric setting, another limitation of the present trial was that it was not fully blinded and thus a different treatment by therapists with possible influence on results cannot be excluded. However, this is not possible due to the different treatments in the two conditions and therefore only patients who had no previous hyperthermia experience were included.Considering the wide range of possible applications of wIRA-WBH, the therapy is perceived to be a good treatment option to reduce pain in patients with FMS. Future research should investigate the effects of mild wIRA-WBH within a multicenter design.


Liquid chromatography coupled with high-resolution mass spectrometry is widely used in composition profiling in untargeted metabolomics research. While retaining complete sample information, mass spectrometry (MS) data naturally have the characteristics of high dimensionality, high complexity, and huge data volume. In mainstream quantification methods, none of the existing methods can perform direct 3D analysis on lossless profile MS signals. All software simplify calculations by dimensionality reduction or lossy grid transformation, ignoring the full 3D signal distribution of MS data and resulting in inaccurate feature detection and quantification.On the basis that the neural network is effective for high-dimensional data analysis and can discover implicit features from large amounts of complex data, in this work, we propose 3D-MSNet, a novel deep learning-based model for untargeted feature extraction. 3D-MSNet performs direct feature detection on 3D MS point clouds as an instance segmentation task. After training on a self-annotated 3D feature dataset, we compared our model with nine popular software  on two metabolomics and one proteomics public benchmark datasets. Our 3D-MSNet model outperformed other software with significant improvement in feature detection and quantification accuracy on all evaluation datasets. Furthermore, 3D-MSNet has high feature extraction robustness and can be widely applied to profile MS data acquired with various high-resolution mass spectrometers with various resolutions.https://github.com/CSi-Studio/3D-MSNet under a permissive license. Benchmark datasets, training dataset, evaluation methods, and results are available at https://doi.org/10.5281/zenodo.6582912.3D-MSNet is an open-source model and is freely available at  Thousands of analytes are eluted at different times and acquired as 3D Gaussian-like features, which are different in volumes and shapes, mixed with noise, and overlap with each other. Due to the high-dimension, high-complexity, and high-throughput nature of mass spectrometry (MS) data, accurate feature extraction is a key challenge in untargeted data interpretation (Untargeted liquid chromatography–mass spectrometry (LC-MS) is widely used in composition profiling in metabolomics and proteomics . In fullretation .m/z dimension is not directly involved in feature discovery. Although the dimensionality reduction in the m/z dimension brings less computational complexity and faster computation speed, the incomplete analysis of the data distribution affects the accuracy of the feature detection results.Many untargeted analytical tools and software packages were developed to extract features accurately from the complex raw data. These methods can be primarily divided into three categories: 2D analysis, 2.5D analysis, and 3D analysis. Most methods analyze MS data dimension by dimension in 2D and 2.5D to reduce the computation time and complexity. 2D methods, such as MS-DIAL , MZmine m/z and RT information simultaneously for better feature detection and quantification. Since the signal points of MS data are sparsely distributed in 3D space with high throughput and complexity, direct analysis of raw MS data is challenging. In the last decades, more and more methods attempted to perform 3D analysis and evaluate the To achieve precise MS data quantification and avoid information loss in data transformation, in this article, we propose a novel method to fully utilize the 3D distribution of MS data, named as 3D-MSNet. 3D-MSNet is a single-stage multi-task deep learning-based model for point-wise 3D feature detection and quantification on profile full-scan and DDA data . InsteadMetabolomics datasets. We used the two datasets published in Proteomics dataset. We used the Orbitrap XL dataset    for 3D-MAs far as we know, no public dataset provides 3D point-wise feature annotation results. We manually annotated a 3D feature instance dataset to train and evaluate our model, named the 3DMS dataset.3DMS dataset. The 3DMS dataset contains 306 point clouds with 1 007 297 annotated signal points and 3727 annotated features. The point clouds were extracted from the first replicate of mixture SA (SampleA_1) in the metabolomics TripleTOF 6600 dataset, with fixed extraction windows at 0.8 Da m/z width and 6 min RT width. For clearer labeling, signals with intensity lower than 128 were considered as noise and not inherited to the extracted point clouds. All point clouds were point-wise labeled using the annotation software from BasicAI (www.basic.ai). Each signal point was assigned an instance label indicating whether the point belonged to a feature, and to which feature it belonged. All annotation results were checked by three experts and can be freely downloaded as .csv files at Zenodo (https://doi.org/10.5281/zenodo.6582912).3D-MSNet supports two raw file formats: mzML and Aird . We used3D-MSNet segments MS data into point cloud blocks for processing . Althougm/z dimension, data are also centered to zero and normalized by the theoretical m/z FWHM. The theoretical m/z FWHM is calculated from three parameters: mass analyzer principle, an m/z value, and the resolution at the m/z. 3D-MSNet supports time-of-flight (TOF) and Orbitrap mass analyzers, which are different in theoretical resolution calculation. All normalization operations are reversible so that the accuracy is maintained.Normalization was performed for better model generalization. On the intensity dimension, base two logarithmic transformation is applied to point intensities, while obvious feature shapes are retained in different abundances. On the RT dimension, data are centered to zero and normalized according to the typical RT full width at half maximum (FWHM). On the 3D-MSNet improves the accuracy of feature detection and quantification by means of direct and complete utilization of raw MS data. The architecture of 3D-MSNet is illustrated in The backbone of 3D-MSNet is well-designed for point-wise hierarchical feature extraction. After data partition and normalization, 3D MS point clouds are fed into the network backbone. To extract hierarchical spatial features, first, the Local Spatial Encoding Block is applied to extract the local spatial features of each point. The Local Spatial Encoding Block uses a shared multilayer perception (MLP) to encode nearest neighbor 3D coordinates to spatial features. Then, multiple Encoding Blocks are applied for hierarchical feature encoding. In each Encoding Block, point features are aggregated by intensity probability sampling and point-wise interpolated convolution. While point clouds are sampled into fewer points, each sampled point obtains higher-level features by aggregating features from a larger receptive field. Next, multiple Decoding Blocks are applied for propagating the extracted hierarchical features to raw MS points. In each Decoding Block, point features abstracted from different levels are combined by nearest-neighbor interpolation and concatenation, and then a shared MLP is applied to transform concatenated features to final decoded features. The detailed implementation methods of Local Spatial Block, Encoding Block, and Decoding Block are illustrated in m/z and RT plane represented by a center point and multiple equiangular rays in Polar Coordinates , PyTorch (version 1.9), CUDA (version 11.1), and cuDNN (version 8.0.5) on Ubuntu (version 16.04) and further trained, validated, and tested on a computer with an Intel(R)_Core(TM)_i9-10900K CPU, 128GB memory, and a GeForce RTX 3090 GPU.We used the self-annotated 3DMS dataset for network training and validation. The 3DMS dataset was randomly separated into the training set and validation set according to the ratio of 7:3. From the 306 annotated point clouds, 214 point clouds were selected for training and the rest 92 point clouds for validation.m/z and RT dimensions were added with random offsets. To improve the training efficiency and stability, in each training epoch, eight randomly selected annotated point clouds were fed to the model simultaneously as a batch. Low-intensity simulated chemical noises were filled alongside the real data for small point clouds to make the number of points consistent within each point cloud of the batch.To improve the model’s generalization ability, data augmentation was applied to the training dataset in the loading stage of every training epoch to increase the data amount and enrich the data diversity. In the training process of 3D-MSNet, data in all dimensions were scaled by a random factor of 0.5 to 2, and data in The loss functions of 3D-MSNet are summarized in To evaluate the performance of 3D-MSNet training, we plotted the loss and accuracy curves of each prediction branch during the training process . As the To further demonstrate that 3D-MSNet did not overfit during training, we evaluated the model pre-trained on the 3DMS dataset on three public benchmark datasets with different data distributions below. 3D-MSNet showed high accuracy on all datasets. The model learned general feature point cloud distributions robustly, which proved that the training was not overfitting.We compared 3D-MSNet with five popular untargeted metabolomics software: MarkerView (version 1.3.1), Compound Discoverer (version 3.2.0.421), MS-DIAL (version 4.70), MZmine 2 (version 2.53), and XCMS Online (version 2.7.2), on both the TripleTOF 6600 dataset and the QE HF dataset in terms of untargeted feature detection, quantification, and discriminating marker selection. Software parameters were fine-tuned to maximize the identification rate and were summarized in m/z and RT tolerances at  on the TripleTOF dataset, and  on the QE HF dataset (https://github.com/CSi-Studio/MetaPro).In evaluation of feature detection and quantification performance, we used the feature libraries provided by the dataset, which contained 970 features in the TripleTOF 6600 dataset and 836 features in the QE HF dataset. Since the standard mixtures only differed in concentration, for each dataset, we first retained the consensus features detected in all samples. Then, the consensus features were matched to the library with  dataset . To eval3D-MSNet achieved the best feature detection and quantification performance on both datasets . In the To evaluate the accuracy of quantification, we compared the measured relative quantification results with the theoretical results. The log fold-changes (SB:SA) of matched features between standard mixtures were divided into seven groups according to the theoretical concentration ratio  and plotted in In evaluation of marker detection performance, we considered the consensus features whose fold-changes were out of 20% tolerance of the range of  as markers 1. True mWe further evaluated 3D-MSNet on the Orbitrap XL dataset against four proteomics software: MaxQuant (version 2.0.3.1), Dinosaur (version 1.2.0), DeepIso, and PointIso. MaxQuant and Dinosaur are 2.5D traditional methods, and DeepIso and PointIso are 3D deep learning methods. We followed the parameter settings of MaxQuant and Dinosaur in the Orbitrap XL dataset paper and used default parameters and models for DeepIso and PointIso as recommended by their author. 3D-MSNet parameters are listed in m/z and RT tolerances used in matching were set to 0.01 Da and 0.4 min according to the match deviation distributions  identifications with peptide score >25 were considered as high-confidence features. ibutions . Featurefeatures 2. 3D-MSNfeatures , and thefeatures . In the features .Then, we used the spike-in and background peptide library to evaluate feature extraction performance under different dilution levels. The library was provided in the targeted selected reaction monitoring (SRM) dataset paired with the Orbitrap XL dataset, which shared the same LC platform and parameters. Since the Orbitrap XL dataset has significant RT and intensity variations in 57 injections, we introduced additional alignment and normalization processing. Limited by main text pages, the analysis process and results are detailed in m/z dimension was caused by the rapid change of the proportion of liquid chromatography solvent. In experiment methods, the proportion of solvent A was rapidly reduced from 65% at 95 min to 10% at 98 min, and huge amounts of analytes were eluted into the mass spectrometer within a short period of time. The central dense region of the feature exhibited a chemical noise-like distribution along the m/z dimension. 3D-MSNet did not detect these anomalous signals as features and performed accurate noise-signal discrimination in all complex scenarios. Among the comparison methods, 3D-MSNet achieved the highest feature detection precision.For a more comprehensive evaluation not limited to selected features, we further evaluated the accuracy of the full feature detection results based on the distribution of the raw MS data. The distribution of untargeted feature detection results varied significantly among different software . We seleThe purpose of 3D-MSNet is to learn the feature distribution robustly and improve untargeted feature detection and quantification accuracy on profile LC coupled with high-resolution MS data. In all the evaluations, 3D-MSNet used the same model pre-trained on the 3DMS dataset. The TripleTOF 6600 dataset was acquired from the same sample set as the training 3DMS dataset by the same LC-MS platform. The QE HF dataset was acquired from the same sample set by a different LC-MS platform, which used a mass detector with different working principles and resulted in different feature shapes, feature volumes, and signal densities. The proteomics Orbitrap XL dataset was acquired from a different sample set and a different LC-MS platform, which resulted in completely different data distribution with the training 3DMS dataset. On all the three datasets, 3D-MSNet outperformed other software in terms of feature extraction and proved its effectiveness and robustness. We also evaluated proteomics software on the metabolomics datasets. The proteomics software detected fewer features than metabolomics software , which fWe further tested the generalization ability of 3D-MSNet on more datasets  with theWe measured the running times of all comparison software on all evaluation datasets . AlthougFeature extraction is fundamental to metabolomics LC-MS data interpretation. The accuracy of feature detection and quantification determines the rationality and credibility of subsequent statistical and biological analysis. In this study, we propose 3D-MSNet to learn the 3D feature distribution robustly and improve untargeted feature detection and quantification accuracy on profile LC-MS data. 3D-MSNet treats the feature extraction task on MS data as an instance segmentation problem on 3D MS point clouds. Using a multitask hierarchical deep neural network, 3D-MSNet enables direct spatial analysis of lossless MS data for the first time. Due to the comprehensive analysis of data distribution, 3D-MSNet showed obvious advantages in signal-noise discrimination and overlapping feature separation. In comparison with popular software, 3D-MSNet made consistent and significant improvements in feature detection and quantification accuracy on all the three metabolomics and proteomics benchmark datasets with competitive computational time.3D-MSNet is a successful attempt to 3D analysis of MS data, providing a new way for more comprehensive MS data analysis. For now, 3D-MSNet only provides command-line-based interaction. In the future, we will develop a supporting graphical interface for 3D-MSNet to facilitate users to generate, check, and correct results.btad195_Supplementary_DataClick here for additional data file.


We prepared a rhodamine-TEMPO chromophore-radical dyad (RB-TEMPO) to study the radical enhanced intersystem crossing (REISC). The visible light-harvesting chromophore rhodamine is connected with the TEMPO  via a C–N bond. The UV-vis absorption spectrum indicates negligible electron interaction between the two units at the ground state. Interestingly, the fluorescence of the rhodamine moiety is strongly quenched in RB-TEMPO, and the fluorescence lifetime of the rhodamine moiety is shortened to 0.29 ns, from the lifetime of 3.17 ns. We attribute this quenching effect to the intramolecular electron spin–spin interaction between the nitroxide radical and the photoexcited rhodamine chromophore. Nanosecond transient absorption spectra confirm the REISC in RB-TEMPO, indicated by the detection of the rhodamine chromophore triplet excited state; the lifetime was determined as 128 ns, which is shorter than the native rhodamine triplet state lifetime (0.58 μs). The zero-field splitting (ZFS) parameters of the triplet state of the chromophore were determined with the pulsed laser excited time-resolved electron paramagnetic resonance (TREPR) spectra. RB-TEMPO was used as a photoinitiator for the photopolymerization of pentaerythritol triacrylate (PETA). These studies are useful for the design of heavy atom-free triplet photosensitizers, the study of the ISC, and the electron spin dynamics of the radical-chromophore systems upon photoexcitation. For the D1 state, however, the chromophore could be in an open-shell electronic configuration . Dn → D1 transition is overall electron spin allowed; thus, the ISC of the chromophore becomes spin allowed. As a result, attachment of a stable radical to a chromophore usually results in the ISC of the light-harvesting chromophore [Triplet photosensitizers (PSs) have attracted much attention in recent years ,2,3,4, bient ISC ,29. Althient ISC ,31. Charient ISC ,33,34. Fplet PSs ,36,37,38plet PSs ,40. In tplet PSs ,42. The omophore ,40,43. Somophore ,44, peryomophore , naphthaHowever, it is clear that much room is left for using this molecular structure protocol to design new heavy atom-free triplet PSs. For instance, the selection of a proper chromophore and the linker between the radical and the chromophore is critical to optimize the photophysical property of the resulted dyad for the purpose of designing an ideal triplet PSs . MoreoveIn order to address the above challenges, herein we prepared a rhodamine-TEMPO . For RB-TEMPO, however, no such absorption band was observed, indicating that the RB-TEMPO molecules adopt a closed ring, lactam structure. TEMPO does not show any strong absorption in the visible spectral range. In order to confirm that RB-TEMPO has a lactam structure, we studied the evolution of the UV-vis absorption spectra of RB-TEMPO in the presence of acid in a protic solvent  than the rhodamine reference compound RBC (k = 1.5 × 10−3 s−1). The UV-Vis absorption spectra of RBC with the addition of TFA is shown in We also studied the kinetics of the transformation of RB-TEMPO from the lactam structure to the opened amide structure, by following the absorbance at 555 nm c. RBC is3•, the quenching mechanism was not studied in detail [The fluorescence property of the compounds was studied . In Figun detail ,50. The 1 state of the rhodamine moiety in RB-TEMPO is attributed to the REISC effect (see later section).The fluorescence lifetimes of the compounds were studied b. For th1 state emit fluorescence before encountering with the TEMPO radical via diffusion in the fluid solution. We also measure the fluorescence lifetime of EY  is much lower than RB (ΦF = 62.7%). As an approximation of the ISC quantum yields, we measured the singlet oxygen (1O2) quantum yields (ΦΔ) of the compounds. RB has no 1O2 photosensitizing ability, and the ΦΔ is null. For RB-TEMPO, however, the ΦΔ was determined as 40.1%. This is attributed to the REISC effect, due to the electron spin–spin interaction between the TEMPO and the photoexcited RB moiety. The raw data for singlet oxygen yield are listed in the The photophysical parameters of the compounds are presented in In order to study the formation of the triplet state of RB moiety in RB-TEMPO, the ns-TA spectra upon pulsed laser photoexcitation of the compound were recorded . A grounThe transient species lifetime was determined as 128 ns b, by monThis result shows that the linker length  should be optimized for designing a heavy atom-free triplet PSs based on the REISC mechanism. Previously, a short triplet state lifetime of perylene was determined as 100 ns in a perylene–nitroxide dyad, which is attributed to the short distance between the nitroxide spin carrier and the perylene chromophore. We show that in Bodipy–TEMPO dyads, the longer the linker is, the longer the triplet state lifetime of the Bodipy moiety may have. However, the ISC quantum yield may be low for dyads containing a long linker .2 = 3/4) and quartet state (square of angular momentum S2 = 15/4) exist. However, the optical spectroscopy, such as the ns-TA spectroscopy, is unable to discriminate the doublet state and the quarter state, because these states share similar energy, and the difference is the electron spin–spin interaction. Moreover, the absorption of the D1 and Q1 state are virtually the same, i.e., the electronic transition to higher states of the chromophore or the TEMPO radical. This scenario is different from the S1 state and T1 state of the same chromophore. In a two-electron spin system, in this case, drastically different excited state absorption spectra can be found for the S1 and T1 states.It should be pointed out that here the ‘triplet’ state refers to the RB chromophore’s electronic configuration. Overall, the photoexcited state of RB-TEMPO is a three-electron spin system; only the doublet  and 530 MHz (0.0177 cm−1)  [We noted that the zero-field splitting (ZFS) eviously . Therefo77 cm−1) , respect17 cm−1) ,63. Prev17 cm−1) .1O2) quantum yield (can be approximated as the ISC yield) than RB; all these factors contribute to the better signal for EY. Upon a pulsed laser excitation, an electron spin polarization (ESP) pattern of Eosin Y was observed. This is the typical feature of the TREPR spectrum of a triplet state produced by the spin–orbit coupling effect. With spectral simulation, the ZFS D and E parameters were determined as −2100 MHz and 618 MHz (D parameter (the E parameter was not reported) [Since the ISC of RB is poor, we studied another xanthene dye that shows efficient ISC, i.e., Eosin Y b. As com 618 MHz . This iseported) . In the In order to study the potential application of the compounds, such as in photopolymerization, we studied the photopolymerization of pentaerythritol triacrylate (PETA), with RB-TEMPO as a photoinitiator  65,66,6,666,67. The photopolymerization of PETA in the presence of RB-TEMPO and NB is presented in Interestingly, we found that the reference compound RB is also active to initiate the photopolymerization of PETA p. Note tIn order to study the intermolecular electron transfer or intramolecular electron transfer, after the ion exchange upon mixing of RB/RB-TEMPO (with NB), the UV-vis absorption spectra of RB and RB-TEMPO in the presence of NB upon photoirradiation were recorded . Upon phΦF) were measured with an absolute photoluminescence quantum yield spectrometer . Fluorescence lifetime was measured with an OB920 luminescence lifetime spectrometer . The synthetic procedure and molecular structural characterization data of the compound, i.e., RB-TEMPO, are collected in the following sections. All compounds used in the experiments and presented in All the chemicals used for the synthesis were analytically pure and were used as received without further purification. UV-vis absorption spectra were obtained on a UV-2550 spectrophotometer . Fluorescence spectra were recorded on an FS5 spectrofluorometer . Fluorescence quantum yields . 4-amino-TEMPO  and triethylamine (2 mL) were added under a N2 atmosphere. After stirring at room temperature for 15 min, the reaction solution was refluxed for 21 h. After the reaction is finished, the reaction mixture is cooled to room temperature. The solvent was removed under reduced pressure. The crude product was purified by column chromatography  to afford a red solid in 10% yield (0.101 g). The 1H NMR spectrum was recorded after treatment of the product with phenylhydrazine, to reduce the nitroxide radical to a hydroxyl structure (no satisfactory NMR spectra can be recorded for the paramagnetic compound). 1H NMR : δ 0.82–0.89 , 1.03 , 1.16 , 1.39 , 1.80 , 2.88  3.35 , 6.42 , 6.87–6.93 , 7.16 , 7.53 , 7.92 . HRMS (EI): calcd for C37H48N4O3+·, m/z 596.3721; found m/z 596.3711.Rhodamine B  was dissolved in dry dichloromethane (50 mL), and then phosphorus oxychloride (1 mL) was added under a N2 for 15 min before measurements, and excited with a nanosecond pulsed laser . The signal was digitized with a Tektronix TDS 3012B oscilloscope and the data were processed with L900 software. .The ns TA spectra were measured on LP980 laser flash photolysis spectrometers . Samples were purged with N2 was removed by a few freeze–pump–thaw cycles. The time-resolved continuous-wave (TR CW) EPR spectral measurements were performed on an X-band EPR Elexsys E-580 spectrometer  at 80 K. Samples were excited by laser pulse at 525 nm with 1 mJ energy per pulse. The data were processed with the EasySpin package based on Matlab [Samples were dissolved in ethanol. On Matlab .2), and the polymerization time was recorded to compare the rate of polymerization.In the experiment, four control experiments were carried out. Monomer PETA (1.2 g); PETA (1.2 g) and photosensitizer (0.07 mg); PETA (1.2 g) and NB (2 mg); PETA (1.2 g), NB (2 mg) and photosensitizer (0.07 mg) were added into de-aerated acetonitrile (0.1 mL), respectively. Then, the mixture was photoirradiated under the unfiltered white light of a xenon lamp (light intensity: 80 mW/cmc = 1.0 × 10–5 M) was added into de-aerated acetonitrile (3 mL) to measure its UV-vis absorption spectrum. Then, the co-initiator NB (2 mg) was added to the RB solution. After each 1 min of xenon lamp photoirradiation (unfiltered white light intensity: 80 mW/cm2), the absorption spectra were measured to monitor the photobleaching in the presence of co-initiator NB. The UV-vis absorption spectra of RB solution in the absence of NB were also photoirradiated by a xenon lamp (unfiltered white light intensity: 80 mW/cm2) and was also measured to study the photo-stability.In the experiments, a set of comparison experiments was carried out. In the first experiment, the RB , in the presence of a co-initiator  tetrabutylammonium tris(3-chloro-4-methylphenyl)hexylborate. Intermolecular and intramolecular (ion exchange reaction exist) electron transfer between the rhodamine chromophore and the co-initiator exist, leading to the production of the alkyl radical, which initiates the radical polymerization of the monomers. These studies are useful for the design of heavy atom-free triplet photosensitizers and study of the ISC and electron spin dynamics of these versatile organic molecules.In summary, in order to study the radical enhanced intersystem crossing (REISC), we prepared a rhodamine-TEMPO chromophore–radical dyad (RB-TEMPO). The visible light-harvesting chromophore rhodamine is connected with the TEMPO  via the C–N bond. The UV-vis absorption spectrum indicates a negligible electron interaction between the two units at the ground state. Interestingly, fluorescence of the rhodamine moiety is strongly quenched in RB-TEMPO, and the fluorescence lifetime of the rhodamine moiety is shortened to 0.29 ns, from that of 3.17 ns of the native rhodamine chromophore. We attribute this quenching effect to the intramolecular electron spin–spin interaction between the nitroxide radical and the photoexcited rhodamine chromophore. This conclusion is supported by the control experiment which shows the intermolecular interaction of the rhodamine and TEMPO leads to a much weaker quenching effect. Nanosecond transient absorption spectra confirm the REISC effect in RB-TEMPO, indicated by the detection of the rhodamine chromophore triplet excited state; the triplet state lifetime was determined as 0.128 ns, which is shorter than the rhodamine triplet state lifetime (0.58 μs). The zero-field splitting (ZFS) parameters of the triplet state of the chromophore were determined with the pulsed laser excited time-resolved electron paramagnetic resonance (TREPR) spectra as


Listeria monocytogenes. Cinnamaldehyde, a natural food preservative, is considered an inhibitor of Agr-mediated QS in L. monocytogenes. However, the exact mechanism by which cinnamaldehyde acts on Agr remains unclear. In this study, we assessed the effects of cinnamaldehyde on the histidine kinase AgrC and the response regulator AgrA in the Agr system. AgrC kinase activity was not influenced by cinnamaldehyde, and binding between AgrC and cinnamaldehyde was not observed when microscale thermophoresis (MST) was performed, indicating that AgrC was not the target of cinnamaldehyde. AgrA is specifically bound to the agr promoter (P2) to activate the transcription of the Agr system. However, AgrA-P2 binding was prevented by cinnamaldehyde. The interaction between cinnamaldehyde and AgrA was further confirmed with MST. Two conserved amino acids, Asn-178 and Arg-179, located in the LytTR DNA-binding domain of AgrA, were identified as the key sites for cinnamaldehyde-AgrA binding by alanine mutagenesis and MST. Coincidentally, Asn-178 was also involved in the AgrA-P2 interaction. Taken together, these results suggest that cinnamaldehyde acts as a competitive inhibitor of AgrA in AgrA-P2 binding, which leads to suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes.The Agr quorum sensing (QS) system is known to contribute to biofilm formation in IMPORTANCEListeria monocytogenes can form biofilms on various food contact surfaces, posing a serious threat to food safety. Biofilm formation of L. monocytogenes is positively regulated by the Agr quorum sensing system. Thus, an alternative strategy for controlling L. monocytogenes biofilms is interfering with the Agr system. Cinnamaldehyde is considered an inhibitor of the L. monocytogenes Agr system; however, its exact mechanism of action is still unclear. Here, we found that AgrA (response regulator), rather than AgrC (histidine kinase), was the target of cinnamaldehyde. The conserved Asn-178 in the LytTR DNA-binding domain of AgrA was involved in cinnamaldehyde-AgrA and AgrA-P2 binding. Therefore, the occupation of Asn-178 by cinnamaldehyde suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. Our findings could provide a better understanding of the mechanism by which cinnamaldehyde inhibits L. monocytogenes biofilm formation. Listeria monocytogenes is a Gram-positive bacterium and can cause listeriosis in humans and animals, with typical clinical manifestations including meningitis, septicemia, spontaneous abortion, and neonatal death  –11. QS i–ytogenes . Some sced to QS , becauseytogenes . Althougrom agrB . The fouectively . Among trylation . Then, trylation . The Agrormation , 16. ThuVibrio spp. . As shown by the results in agrBD, agrC, and agrA were significantly decreased (P < 0.05) in the presence of cinnamaldehyde. Additionally, the effects of cinnamaldehyde on the activity of the agr promoter P2 were investigated. Cinnamaldehyde at 1/8 MIC, 1/4 MIC, and 1/2 MIC reduced P2 activity by 17.6, 30.7, and 45.9%, respectively  of AgrC using the TMHMM-2.0 program . AgrC of L. monocytogenes contains two distinct domains: an N-terminal sensor module (residues 1 to 206) and a C-terminal cytoplasmic histidine kinase domain (residues 207 to 431). According to the prediction results of TMHMM, the sensor module comprises six TMSs: residues 2 to 22 (in-to-out), residues 32 to 66 (out-to-in), residues 73 to 95 (in-to-out), residues 110 to 132 (out-to-in), residues 152 to 174 (in-to-out), and residues 184 to 206 (out-to-in) (Since AgrC in t-to-in) . AgrC cot-to-in) . The hisCyto  and AgrCFL (the full-length protein of AgrC) were expressed in the present study. Purified AgrCCyto and AgrCFL were loaded onto SDS-PAGE gels for analysis, and strong bands with molecular masses of approximately 25 and 48 kDa, respectively, were observed (Cyto (residues 207 to 431) has two subdomains, a membrane-proximal dimerization and histidine phosphotransfer (DHp) subdomain and the catalytic and ATP binding (CA) subdomain, which can bind and hydrolyze ATP and autophosphorylate. The kinase activity of AgrCCyto was assayed in our study, and it was inversely related to the residual amount of ATP. In the reaction system, the initial concentration of ATP was 10 μM. After incubation, the remaining ATP was reduced with increasing concentrations of AgrCCyto  was performed to confirm the interaction between cinnamaldehyde and AgrC. As shown by the results in een them . TherefoagrA mutation on agr operon transcription, an ΔagrA gene deletion mutant was constructed in this study. As shown by the results in agrBD and agrC in the ΔagrA mutant were significantly lower (P < 0.05) than those of the wild-type strain EGD-e, and complementation of the agrA mutation restored the transcription of agrBD and agrC. In addition, the activity of the agr promoter P2 in the ΔagrA mutant was clearly decreased (P < 0.05) (To investigate the effect of  < 0.05) . These d2 promoter. Shifted bands of protein-DNA complexes were observed in the gel, indicating binding of the recombinant AgrA protein to the P2 promoter  of 55.93 ± 2.38 (mean ± standard deviation)  , suggestL. monocytogenes EGD-e comprises 242 amino acids and belongs to the LytTR family of response regulators. The AgrA of L. monocytogenes EGD-e shares 42% identity with the AgrA of S. aureus N315 , AgrAD158A, AgrAD159A, AgrAG176A, and AgrAK177A showed binding abilities to cinnamaldehyde similar to that of wild-type AgrA. Compared with that of cinnamaldehyde-AgrA, higher dK values were observed in cinnamaldehyde-AgrAN178A and cinnamaldehyde-AgrAR179A, suggesting lower binding abilities of AgrAN178A and AgrAR179A to cinnamaldehyde  in biofilm biomass was observed when the strain was exposed to 1/8 MIC of cinnamaldehyde, while cinnamaldehyde at concentrations of 1/4 MIC and 1/2 MIC obviously reduced (P < 0.05) biofilm biomass, by 26.1 and 47.2%, respectively. Compared with the control , the addition of cinnamaldehyde did not affect the viability of the planktonic cells , self-phosphorylation of AgrCCyto can be inhibited. However, our results showed that cinnamaldehyde had no effect on the phosphorylation level of AgrCCyto. Furthermore, the results from MST provided direct evidence that cinnamaldehyde did not bind to AgrCCyto. To determine whether cinnamaldehyde binds to the sensor domain of AgrC, the full-length protein, AgrCFL, was expressed and purified. However, binding between cinnamaldehyde and AgrCFL was not observed in this study, suggesting that cinnamaldehyde is not a competitive inhibitor of AgrC-AIP binding. The results seem reasonable, because most of the reported inhibitors of the Agr QS system in S. aureus that can bind to AgrC are peptide mimetics of the native AIP  at the C-terminal end. In our study, cinnamaldehyde appeared to target the AgrA of L. monocytogenes and inhibit AgrA-P2 binding. Therefore, it is very likely that cinnamaldehyde can bind to the LytTR domain of AgrA. The predicted results from molecular docking demonstrated that seven amino acids of AgrA were involved in cinnamaldehyde binding. Except for His-29, the other six sites were located within the LytTR domain. The importance of conserved Asn-178 and Arg-179 in cinnamaldehyde-AgrA interactions was confirmed by alanine mutagenesis and subsequent MST. Furthermore, only Asn-178 was a key site for Agr-P2 binding. Cinnamaldehyde occupies Asn-178, leading to the decreased P2-binding ability of AgrA. Thus, our data indicate that cinnamaldehyde acts as a competitive inhibitor of AgrA-P2 binding. Notably, Asn-178 and Agr-179 are also highly conserved among strains of S. aureus, suggesting an interaction between cinnamaldehyde and AgrA in S. aureus.Several small-molecule compounds, such as bumetanide  and F12 oter DNA , 40. Agroter DNA –43. The L. monocytogenes was proposed. Cinnamaldehyde binds to the LytTR DNA-binding domain of AgrA, which prevents AgrA from binding to the P2 promoter and then inhibits the Agr system. However, several important questions about this mechanism remain answered. One is whether cinnamaldehyde affects AgrA phosphorylation. The AgrA of L. monocytogenes has an N-terminal phosphoacceptor receiver domain, in addition to the LytTR domain. The receiver domain transfers the phosphate group of phosphorylated AgrC to the Asp residue of AgrA and then catalyzes the autophosphorylation of AgrA , while Todd et al. (L. monocytogenes EGD-e) was used in these two studies. Therefore, the possibility that different structures of AIP resulted from strain differences was excluded. In addition, it is likely that L. monocytogenes cells produce a very small amount of AIP, because peptide signaling molecules are more metabolically costly, and their concentrations in bacterial culture are much lower than other types of signaling molecules  broth at 37°C. The strains and plasmids used in this study are presented in Table S1 in the supplemental material.Wild-type L. monocytogenes EGD-e  was prepared by dissolving cinnamaldehyde in ethanol. The agar dilution method was used to determine the MIC of cinnamaldehyde against es EGD-e .L. monocytogenes EGD-e was grown in BHI medium with or without cinnamaldehyde (1/2 MIC). 16S rRNA was used as a reference gene, and the fold changes of the target genes were calculated using the cycle threshold (2−ΔΔCT) method . Primers for QS genes and motility-associated genes have been reported in our previous studies . L. mono) method .agr promoter (P2)-lacZ fusion was constructed as described previously . A β-galactosidase activity assay based on the method of Miller was performed as described previously . The three-dimensional models of AgrA were generated by SwissModel (https://swissmodel.expasy.org/). The docking simulations were performed using AutoDock 1.5.6 tools (https://autodock.scripps.edu/), and PyMOL 2.3.0 (https://pymol.org/2/) was used to analyze the docking results.The chemical structure of cinnamaldehyde was downloaded from the PubChem database  and AgrA (the entire protein) (Table S2). The expression and purification of AgrCFL were performed as described previously . IPTG  was added to bacterial cultures with a final concentration of 0.1 mM to induce the expression of AgrCFL, and the cultures incubated for 24 h at 20°C. After centrifugation, the precipitate was resuspended in phosphate-buffered saline (PBS) containing 1 mM MgCl2, 1 mM protease inhibitor Pefabloc SC (Roche), 100 U/mL DNase, and 20 mM imidazole. Resuspended cells were lysed using ultrasonic waves and then centrifuged. Supernatants were centrifuged at 300,000 × g for 1 h at 4°C. The precipitates were resuspended with PBS containing 10× carboxymethyl cellulose (CMC) and 10 mM imidazole and then incubated on ice with shaking overnight. After centrifugation, supernatants containing the target protein AgrCFL were collected. AgrC was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The purified AgrCFL was analyzed via SDS-PAGE.PCR primers were designed to obtain expression constructs for AgrC (the cytoplasmic domain [residues 207 to 431] is referred to as AgrCeviously . The fulCyto and AgrA were expressed and purified according to the protocols described in our previous study (E. coli BL21(DE3) competent cells. The recombinant protein was expressed by adding IPTG and purified using BeyoGold His-tag purification resin .AgrCus study . BrieflyCyto and AgrCFL kinase activities were evaluated using a Kinase-Lumi chemiluminescence kinase activity detection kit (Beyotime) according to the manufacturer’s instructions. Briefly, the purified protein was incubated with the reaction buffer for 5 min at 37°C, and then ATP was added. After incubation for 10 min, the chemiluminescence of the samples was detected using a CLARIOstar multimode microplate reader . In the kinase assay, the amount of ATP remaining in the solution following a kinase reaction was measured, and luminescence was inversely related to kinase activity. To investigate the effect of cinnamaldehyde on AgrCCyto and AgrCFL kinase activities, cinnamaldehyde at different concentrations was added to the mixture of protein and the reaction buffer, and then the chemiluminescence assay was performed as described above.AgrCdK) were analyzed using the MO.Control Analysis software (NanoTemper).The interaction between cinnamaldehyde and proteins was detected by MST using a Monolith NT.115 instrument  according to the manufacturer’s instructions. Purified proteins were labeled using a protein-labeling kit (NanoTemper). Serial dilutions of cinnamaldehyde were prepared. The labeled protein was incubated with cinnamaldehyde at different concentrations. The samples were then filled into premium capillaries (NanoTemper), followed by measurements taken at 22°C. The interaction affinity and dissociation constant . Plasmid pERL3 was used for complementation experiments as described previously  for 20 min at room temperature. To investigate the effect of cinnamaldehyde on the interaction between the PTo identify the predicted active sites of AgrA, mutants were generated using the QuickMutation site-directed gene mutagenesis kit (Beyotime). The original vector, pET-28a-AgrA, was used as a template for PCR amplification. The primers are listed in Table S2. The nonmutated plasmid template was removed via digestion with DpnI for 1 h at 37°C. The expected mutants were confirmed by direct DNA sequencing. The mutant proteins were expressed and purified as described above.L. monocytogenes biofilms. Biofilms were incubated in 96-well polystyrene plates  and stained with crystal violet as described previously  was carried out. The kinase activity assay results were analyzed using a two-way ANOVA model. Differences with